This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Fanconi anemia (FA) is a rare recessive disorder characterized by congenital anomalies, bone marrow failure, and pronounced cancer susceptibility. FA patient cells are hypersensitive to DNA crosslinking agents such as mitomycin C and cisplatin. The endogenous function of the FA pathway, however, remains unknown. We have recently demonstrated that the FA pathway is functionally required for the cellular response to disruption of DNA replication. The FA pathway is strongly activated via the mono-ubiquitination of the FANCD2 protein following treatment with the DNA polymerase inhibitor aphidicolin (APH). Furthermore, abrogation of the FA pathway leads to increased chromosome breakage, including breakage at the common chromosomal fragile sites FRA3B and FRA16D, following treatment with APH. We hypothesize that the FA pathway plays an integral role in the maintenance of DNA replication fork stability, a function critical for the prevention of neoplastic transformation. Two aims are proposed to gain further insight into this function. First, we will characterize the interaction between the FANCD2 protein and the major DNA polymerase processivity factor PCNA. Specifically, we will examine the functional consequences of mutation of a recently identified FANCD2 PCNA-interaction motif. Second, we will determine if the increased APH-induced chromosome breakage in FA cells leads to increased integration of foreign DNA. Finally, we will attempt to establish a clinical corollary for a role of the FA pathway in the in the maintenance of DNA replication fork stability: We will determine if the increased common chromosomal fragile site breakage of FA cells is associated with an increased frequency of human papillomavirus (HPV) DNA integration at these loci, by mapping the genomic sites of HPV integration in FA patient tumors. Our proposed studies will lead to new findings regarding the in vivo physiological role of the FA pathway, and provide insight into the increased cancer susceptibility of FA patients.